Nb100 56101

The spinal cord tissues of each group were fixed with 4% paraformaldehyde at 4 °C. The spinal cord tissue was placed at the bottom of 50 mL centrifuge tube and dehydrated in 10%, 20%, and 30% sucrose solution. Twelve micrometers frozen sections were made and detected. All sections were blocked with blocking solution [10% goat serum, 3% bovine serum albumin (BSA), and 0.1% Triton X-100] for 1 h at 37 °C and then incubated overnight at 4 °C with Rabbit-anti-Active-Caspase3 (1:500, ab49822, Abcam), Rabbit-anti-Caspase3 (1:500, ab184787, Abcam), Rabbit-anti-LC3 (1:100, ab128025, Abcam), Rabbit-anti-LAMP2 (1:400, L0668, Sigma), Rabbit-anti-Bcl-2 (1:500, NB100-56101, NOVUS) and Rabbit-anti-Bax (1:500, ab12503, Abcam); 0.01 M phosphate buffer saline (PBS) was used to wash them for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again with PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope (Leica DM 5000B, Germany).

The extracted protein was quantified by bicinchoninic acid (BCA) analysis, followed by electrophoresis separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA), the membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS, pH 7.4) and incubated with Rabbit-anti-Caspase 3 (1:1,000, ab184787, Abcam, Cambridge, MA, USA), Rabbit-anti-active-Caspase 3 (1:1,000, ab49822, Abcam), Rabbit-anti-Bax (1:1,000, ab12503, Abcam), Rabbit-anti-Bcl2 (1:1,000, NB100-56101, NOVUS), Rabbit-anti-LC3 (1:500, ab128025, Abcam), Rabbit-anti-TNFR1 (1:1000, 21574-1-AP, Proteintech, Rosemont, IL, USA), Rabbit-anti-TNFR2 (1:1,000, 19272-1-AP, Proteintech), Rabbit-anti-p-Akt (1:1,000, 8200S, Cell Signaling Technology, Danvers, MA, USA), Rabbit-anti-Akt (1:1,000, C67E7, Cell Signaling Technology), Mouse-anti-β-actin (1:5,000, A1978, Sigma-Aldrich, St. Louis, MO, USA), and Rabbit-anti-LAMP2 (1:1,500, L0668, Sigma), respectively, at 4 °C overnight. After being washed with TBST (TBS with 0.1% Tween 20) and incubated with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, the protein was visualized using Beyo ECL Star (Beyotime, Jiangsu, China).